臺灣博碩士論文加值系統

中文摘要
本實驗以黃水茄的下胚軸為培植體，誘導細胞生成固醇類生物鹼，於B5為基本鹽類添加NAA及BA的培養基可誘得生長快速的癒合組織細胞。在液態懸浮培養中，細胞呈現兩階段式的生長，以B5為基本鹽類添加picloram及BA的液態培養基培養細胞可獲得最高的solasodine產量（13.91 mg/g.DW）。在液態懸浮培養中加入asparagine、serine、glycine或yeast extract皆可促進solasodine在細胞中的累積量。
利用Super Spinner Bioreactor（1 L）進行放大培養，以攪拌式半透膜的通氣方式可獲得較佳的細胞增殖量；然後以兩階段式的培養誘導細胞生產solasodine，培養12天後，細胞量由原先50 g增加為101.52 g，在第二階段solasodine於細胞中的含量高達6.88 mg/g.DW。

ABSTRACT
Fast growing calli were induced from hypocotyl explant of Solanum incanum on B5 basal medium contained with NAA plus BA. In cell suspension culture, the growth curve of cell displayed two growth phases. Cell cultured in the B5 basal medium contained with picloram plus BA showed the highest solasodine accumulation（13.91 mg/g.DW）within 12 days. Addition asparagine、serine、glycine or yeast extract to liquid suspension medium can promote solasodine accumulation in cell.
Cell was scaled up by Super Spinner bioreactor（1L）. Two ways of aeration: ventilated gas with stirred permeability membrane and air lift were tested. The former was better than the latter in cell multiplication. For two stages culture, fresh weight of cell was increased from 50 g to 101.52 g in the first stage（6 days）and the accumulation of solasodine in cell was 6.88 mg/g.DW in the secondary stage（6 days）.

目錄
英文摘要……………………………………………………………….....i
中文摘要………………………………………………………………....ii
List of Tables……………………………………………………….…vii
List of Figures……………………………………………………….…iv
縮寫字………………………………………………………..……...xviii
一、 前人研究………………………………………………………....1
二、 材料與方法……………………………………………………..11
2.1材料來源………………………………………………………….....11
2.2培養基…………………………………………………………….....11
2.3培養環境………………………………………………………….....11
2.4癒合組織之誘導………………………………………………….....11
2.5癒合組織的誘導測試…………………………………………….....14
2.6癒合組織之維持………………………………………………….....14
2.7細胞懸浮培養之建立及維持……………………………….…......17
2.8細胞生長曲線及不同條件處理對細胞生長及固醇類生物鹼生產之影響………………………………………….………..………...........17
2.8.1光照培養細胞之生長曲線………………………………….......17
2.8.2 黑暗培養細胞之生長曲線…………………………………......17
2.8.3 接種密度之對細胞生長及固醇類生物鹼生產的影響……......19
2.8.4 植物生長調節劑對細胞生長及固醇類生物鹼生產的影響…………………………………………………………..............19
2.8.5 蔗糖含量對細胞生長之影響……………………………........19
2.8.6 添加物對細胞生長及固醇類生物鹼生產之影響………........19
2.8.7 饋入不同的碳水化合物對細胞生長及固醇類生物鹼生產之影響……………………………………….………………...............20
2.9田間作物固醇類生物鹼的含量……………………..………..…...20
2.10 利用Super Spinner Bioreactor進行批次培養……………..….20
2.11 利用Super Spinner Bioreactor進行二階段培養…………..….21
2.12 細胞鮮重和乾重的測量……………………………………….....21
2.13 固醇類生物鹼的萃取………………………………………….....21
2.14 固醇類生物鹼的分析……………………………………….…....23
2.15 碳水化合物之分析…………………………………………….....23
2.16 Solasodine和Solamargine標準品………………………….....23
2.17 碳水化合物標準品…………………………………………….....23
2.18 型態觀察及照相……………………………………………….....24
2.19 統計分析……………………………………………………….....24
三、 結果……………………………………………………………....28
3.1 癒合組織的誘導………………………………………………....28
3.2細胞生長曲線及不同培養條件處理對細胞生長與固醇類生物鹼生產之影響…………………......…………………..……..............32
3.2.1光照培養細胞之生長曲線…………………………….…...32
3.2.2 黑暗培養細胞之生長曲線………………………………........32
3.2.3 接種量對細胞生長及固醇類生物鹼生產的影響………........33
3.2.4 蔗糖濃度對細胞生長及固醇類生物鹼生產之影響….....33
3.2.5 植物生長調節劑對細胞生長及固醇類生物鹼生產之影響…………………………………………………………..............33
3.2.5.1 2,4-D對細胞生長及固醇類生物鹼生產之影響……..33
3.2.5.2 IAA對細胞生長及固醇類生物鹼生產之影響…….….39
3.2.5.3 Picloram對細胞生長及固醇類生物鹼生產之影響.…39
3.2.5.4 TDZ對細胞生長及固醇類生物鹼生產之影響…..…..39
3.2.6 添加物對細胞生長及固醇類生物鹼生產之影響……...…40
3.2.6.1 asparagine對細胞生長及固醇類生物鹼生產之影響..40
3.2.6.2 glycine對細胞生長及固醇類生物鹼生產之影響……40
3.2.6.3 serine對細胞生長及固醇類生物鹼生產之影響…..…40
3.2.6.4 yeast extract對細胞生長及固醇類生物鹼生產之影響………………………………………………….……................45
3.2.7饋入不同的碳水化合物對細胞生長及固醇類生物鹼生產之影響…………………………………………….…………................45
3.3 田間作物中固醇類生物鹼的含量………………………….......…45
3.4 Super Spinner bioreactor批次培養……………………..…….45
3.5 Super Spinner bioreactor兩階段式培養………………………50
四、 討論……………………………….……………………………....54
4.1 癒合組織的誘導……………………………………………..…...54
4.2 細胞生長曲線測試………………………………………..……...55
4.3 細胞接種量對細胞生長及固醇類生物鹼生產的影響……….....56
4.4 蔗糖含量對細胞生長之影響…………………..…….…………..56
4.5 植物生長調節劑對細胞生長及固醇類生物鹼生產之影響….....56
4.6 添加物對細胞生長及固醇類生物鹼生產之影響………….…....57
4.7 饋入不同的碳水化合物對細胞生長及固醇類生物鹼生產之影響…………………………………………...…………….….…........57
4.8田間作物固醇類生物鹼的含量…………….………….………....58
4.9 Super Spinner bioreactor批次培養…………………...…………58
4.10 Super Spinner bioreactor兩階段式培養………………………59
五、 結論…………………………………………………………..…..61
六、 參考文獻……………………………………………………….....63

參考文獻
林松原, 1986. 台灣產茄科植物成分研究─黃水茄成分探討. 碩士論文. 私立高雄醫學院藥學研究所. pp.12-16
施淑女, 1995. 錢銅樹之三萜類分析＆黃水茄的細胞培養及其二次代謝物. 碩士論文. 國立台灣醫學院藥學研究所. pp.73-78
許淑慧, 1997. 台灣產黃水茄成分中Solamargine之誘發細胞進行設定性死亡及其抗癌作用之分子機制的研究. 博士論文. 私立高雄醫學院藥學研究所. pp.1-12
顏金鳳, 1989. 台灣產茄屬植物及毛葉冬珊瑚植物的保肝及抗腫瘤活性成分的研究. 碩士論文. 私立高雄醫學院藥學研究所. pp.1-4
Ahkam Subroto M. A. and P. M. Doran, 1994. Production of Steroidal alkaloids by Hairy Roots of Solanum aviculare and the Effect of Gibberellic Acid, Plant Cell, Tissue and Organ Culture. 38:93-102
Alvarez M. A., J. R. Talou, N. B. Paniego and A. M. Giulietti, 1994. Solasodine Production in Transformed Organ Culture(Roots and Shoots) of Solanum eleagnifolium Cav., Biotechnology Letters. 16:393-396
Barnabas N. J. and S. B. David, 1988. Solasodine Production by Immobilized Cells and Suspension Culture of Solanum surattense, Biotechnology Letters. 10:593-596
Beaman-Mbaya V. and S.I. Muhammed, 1976. Antibiotic Action of Solanum incanum Linnaeus, Antimicrobial Agents and Chemotherapy. pp920-924
Bhatt P. N., D. P. Bhatt and I. Sussex, 1983. Studies on Some Factors Affecting Solasodine Contents in Tissue Cultures of Solanum nigrum, Physiol. Plant. 57:159-162
Cham B. E., M. Gilliver and L. Wilson, 1987. Antitumour Effects of Glycoalkaloids Isolated from Solanum sodomaeum, Planta Med. 54:34-36
Cham B. E. and B. Daunter, 1990. Solasodine Glycosides. Selective Cytotoxicity for Cancer Cells and Inhibition of Cytotoxicity by Rhamnose in Mice with Sarcoma 180, Cancer Letters. 55:221-225
Cham B. E., R. A. Evans and B. Daunter, 1991. Topical Treament of Malignant and Premalignant Skin Lesions by Very Low Concentractions of a Standard Mixture (BEC) of Solasodine Glycosides, Cancer Letters. 59:183-192
Chandler S. F. and J. H. Dodds, 1983a. Solasodine Production in Rapidly Proliferating Tissue Cultures of Solanum laciniatum Ait., Plant Cell Rep. 2:69-72
Chandler S. F. and J. H. Dodds, 1983b. The Effect of Phosphate, Nitrogen and Sucrose on the Production of Phenolics and Solasodine in Callus Cultures of Solanum laciniatum, Plant Cell Rep. 2:205-208
Chataing B., J. L. Concepcion, R. Lobaton and A. Usubillaga, 1998. Inhibition of Trypanosoma Cruzi Growth In Vitro by Solanum Alkaloids:A Comparison with Ketoconazole, Planta Med. 64:31-36
Daunter B. and B. E. Cham, 1990. Solasodine Glycosides. In Vitro Preferential Cytotoxicity for Human Cancer Cell, Cancer Letters. 55:209-220
Drewes F. E. and J. V. Staden, 1995. Initiation of and Solasodine Production in Hairy Root Cultures of Solanum mauritianum Scop., Plant Growth Regulation. 17:27-31
Duncan D. B., 1955. Multiple Range and Multiple F-test. Biometrics. 11:1-42
Ehmke A., D. Ohmstede and U. Eilert, 1995. Steroidal Glycoalkaloids in Cell and Shoot Teratoma Cultures of Solanum dulcamara, Plant Cell, Tissue and Organ Culture. 43:191-197
El-Badaoui H., B. Muguet and M. Henry, 1996a. Production of Solamargine by In Vitro Culture of Solanum paludosum, Plant Cell, Tissue and Organ Culture. 45:123-127
El-Badaoui H., P. Morard and M. Henry, 1996b. Stimulation of the Growth and Solamargine Production by Solanum paludosum Multiple Shoot Cultures Using a New Culture Medium, Plant Cell, Tissue and Organ Culture. 45:153-158
Eltayeb E. A., A. S. Al-Ansari and J. G. Roddicl, 1997. Changes in the Steroidal Alkaloids Solasodine During Development of Solanum nigrum and Solanum incanum, Phytochemistry. 46:489-494
Gamborg O. L., R. A. Miller and K. Ojima, 1968. Nutrient Requirements of Suspension Culture of Soybean Root Cells. Exp. Cell Res. 50:151-158
Gan K. H. and C. N. Lin, 1993. Cytotoxic Principles and Their Derivatives of Fromosan Solanum Plants, J. Nat. Prod. 56:15-21
Hsu S.-H., T.-R. Tsai, C.-N. Lin, M.-H. Yen, and K.-W. Kuo, 1996. Solamargine Purified from Solanum incanum Chinese Herb Triggers Gene Expression of Human TNFR1 which May Lead to Cell Apoptosis, Biochemical and Biophy. Res. Comm. 229:1-5
Kittipongpatana, N. , R. S. Hock and J. R. Porter, 1998. Production of Solasodine by Hairy Root, Callus and Cell Suspension Culture of Solanum aviculare Forst, Plant Cell, Tissue and Organ Culture. 52:133-143.
Kuo K. W., S. H. Hsu, Y. P. Li, W. L. Lin, L. F. Liu, L. C. Chang, C. C. Lin, C. N. Lin and H. M. Sheu, 2000. Anticancer Activity Evaluation of the Solanum Glycoalkaloid Solamargine, Biochem. Pharm. 60:1865-1873
Lee K.-H., Y.-M. Lin, T.-S. Wu, D.-C. Zhang, T. Yamagishi, T. Hayashi, J.-J Chang, R.-Y. Wu, and T.-H. Yang, 1988. The Cytotoxic Principles of Prunella vulgaris, Psychortria Serpens and Hyptis capitata:Ursolic Acid and Related Derivatives. Plant Med. 54:308-310
Lin C.-N., C.-M. Lu, M.-K. Cheng and K.-H Gan, 1990. The Cytotoxic Principles of Solanum incanum, J. Nat. Prod. 53:513-516
Lin C.-H., M.-I. Chung and K.-H. Gan, 1988. Novel Antihepatotoxic Principles of Solanum incanum, Plant Med. 54:222
Mak Y. and P. M. Doran, 1993. Effect of Cell-Cycle Inhibition on Synthesis of Steroidal Alkaloids by Solanum aviculare Plant Cells, Biotech. Letters. 15:1031-1034
Malpathak N. P. and S. B. David, 1992. Stimulation of Solasodine Production by Combining Fungal Elicitors and Immobilized Cell Suspension Cultures of Solanum surattense Burm, Biotech. Letters. 14:965-968
Mann J. D, 1978. Production of Solasodine for the Pharmaceutical Industry, Adv. in Agron. 30:207-245
Murashige T., F. Skoog, 1962. A Revised Medium for Rapid Growth and Bioassays with Tabacco Tissue Culture. Physiol. Plant. 15:473-497
Nigra H. M., O. H. Caso and A. M. Giulietti, 1987. Production of Solasodine by Calli from Different Parts of Solanum eleagnifolium Cav. Plant, Plant Cell Rep. 6:135-137
Nigra H. M., M. A. Alvarez and A. M. Giulietti, 1989. The Influence of Auxins, Light and Cell Differentiation on Solasodine Production by Solanum eleagnifolium Cav. Calli, Plant Cell Rep. 8 : 230-233.
Rowan D. D., P. E. Macdonald and R. A. Skipp, 1983. Antifungal Stress Metabolites from Solanum aviculare, Phytochemistry. 22:2102-2104
Scragg A. H., 1999. Chapter 36 : Alkaloid Accumulation in Catharanthus roseus Suspension Cultures, In : R. D. Hall edited, Methods in Molecular Biology. Humana Press Inc., Totowa, NJ. pp.393-340
Saijo R., K. Murakami and T. Nohara , 1982. Studies on the Constituents of Solanum Plants. II. Yakugaku Zosshi. 102:300
Weissenberg M., A. Levy, J. A. Svoboda and I. Ishaaya, 1998. The Effect of Some Solanum Steroidal Alkaloids and Glycoalkaloids on Larvae of the Red Flour Beetle, Tribolium Castaneum, and the Tobacco Hornworm, Manduca Sexta, Phytochemistry. 47:203-209